Leukemogenesis as a new approach to investigate the correlation between up regulated gene 4/upregulator of cell proliferation (URG4/URGCP) and signal transduction genes in leukemia.

The aim of the study is to the determine the profiles of cell cycle genes and a new candidate oncogene of URG4/URGCP which play role in leukemia, establishing the association between the early prognosis of cancer and the quantitation of genetic changes, and bringing a molecular approach to definite diagnosis. In this study, 36 newly diagnosed patients' with ALL-AML in the range of 0-18 years and six control group patients' bone marrow samples were included. Total RNA was isolated from samples and then complementary DNA synthesis was performed. The obtained cDNAs have been installed 96 well plates after prepared appropriate mixtures and assessed with LightCycler(®) 480 Real-Time PCR quantitatively. CHEK1, URG4/URGCP, CCNG1, CCNC, CDC16, KRAS, CDKN2D genes in the T-ALL group; CCND2, ATM, CDK8, CHEK1, TP53, CHEK2, CCNG2, CDK4, CDKN2A, E2F4, CCNC, KRAS genes in the precursor B-ALL group and CCND2, CDK6 genes in the AML group have shown significant increase in mRNA expression level. In the featured role of acute leukemia the regulating signaling pathways of leukemogenesis partially defined, although identification of new genetic markers in acute leukemia subgroups, will allow the development of early diagnostic and new treatment protocols.

Molecular Evaluation of t(14;18)(bcl-2/IgH) Translocation in Follicular Lymphoma at Diagnosis Using Paraffin-Embedded Tissue Sections.

OBJECTIVE: Follicular lymphoma (FL) is one of the most common lymphomas, and is characterized by t(14;18)(q32;q21) in more than 80% of patients. The aim of this study was to determine the rate of t(14;18) positivity based onthe detection of mbr or mcr in paraffin-embedded tissue samples. MATERIAL AND METHODS: The study included 32 paraffin-embedded tissue samples collected from 32 consecutive FL patients that were diagnosed and followed-up at our hospital between 1999 and 2006. The MBR breakpoint wasidentified based on real-time PCR using a LightCycler v.2.0 t(14;18) Quantification Kit (MBR), multiplex PCR, and seminestedPCR. To identify the mcr breakpoint, real-time PCR was performed using specific primers and the FastStart DNAMaster SYBR Green I Kit. To detect t(14;18) via fluorescence in situ hybridization (FISH) nuclei from paraffin-embeddedtissue sections were extracted and used together with LSI IgH (immunoglobulin heavy chain) (spectrum green)/bcl-2(B-cell leukemia-lymphoma 2) (spectrum orange) probes. RESULTS: The DNA and nuclei isolation success rate for B5 formalin-fixed, paraffin-embedded tissue sections (n = 12)was 42% and 33%, respectively, versus 95% and 60%, respectively, for 20 tissue sections fixed in formalin only. In all,24 paraffin-embedded tissue sections were analyzed and mbr positivity was observed in the DNA of 82.14% via seminested PCR, in 53.57% via multiplex PCR, and in 28.57% via real-time PCR. We did not detect mcr rearrangementin any of the samples. In all, 15 of 16 patients (93.75%) whose nuclei were successfully isolated were observed to bet(14;18) positive via the FISH method. CONCLUSION: Semi-nested PCR and FISH facilitated the genetic characterization of FL tumors. As such, FISH and PCR complement each other and are both essential for detecting t(14;18) translocation.

Determining the relation between N-acetyltransferase-2 acetylator phenotype and antituberculosis drug induced hepatitis by molecular biologic tests.

Tuberculosis is treated with a group of drugs that need to be used over a long period of time and isoniazid is the major drug in this group. Antituberculosis drug-induced hepatitis is the most serious problem in tuberculosis treatment. The enzyme N-acetyltransferase-2 (NAT-2) metabolizes isoniazid in the liver so it is considered to cause hepatotoxicity. The association of polymorphic NAT acetylator status and antituberculosis drug-induced hepatitis is discussed. To determine whether acetylator status is a risk factor for antituberculosis drug-induced hepatitis, we genotyped NAT2*5A, NAT2*6A, NAT2*7A/B and NAT2*14A polymorphisms in 100 patients diagnosed with tuberculosis. 70 patients who did not develop hepatotoxicity were classified as the control group, and 30 patients who were diagnosed with antituberculosis drug-induced hepatitis were classified as the study group. NAT2 polymorphisms were divided into three phenotypic groups according to the analytical results obtained. Among the 70 patients constituting the control group; 14 (20%), 37 (52.9%), 19 (27.10%) patients were rapid, intermediate and slow acetylators respectively. In contrast, among the patients constituting the study group; 3 (10%), 4 (13.3%), 23 (76.7%) patients were rapid, intermediate and slow acetylators. The difference was statistically significant when the control and study groups were compared for their acetylator status. The proportion of slow acetylators was much higher in the study group. In conclusion, NAT2 acetylator phenotype analysis by molecular biology methods prior to medical treatment for tuberculosis, can be used both for determining the high-risk group of patients who may develop hepatotoxicity and for closer follow-up during treatment period.

The role of angiotensin-converting enzyme and apolipoprotein-E gene polymorphisms on lipid compositions in newborn infants with intrauterine growth restriction.

Recent findings suggest that hypertension, dyslipidemia, diabetes mellitus, coronary heart disease are more common in adults who born with intrauterine growth restriction (IUGR). Several studies have shown that polymorphisms in angiotensin-converting enzyme (ACE) and apolipoprotein-E (Apo-E) are effective in developing the insulin resistance and also in increasing the risk of coronary heart disease. In present study, the frequencies of ACE, Apo-E gene polymorphisms, apolipoprotein-B (Apo-B) mutation and lipid compositions were determined in full-term newborn infants with IUGR. Forty-four newborn infants who had completed 36 weeks of gestational age, 24 healthy infants and 20 with IUGR, were taken into the scope of the study. While total cholesterol (TC) and Apo-B concentrations in infants with IUGR was found to be significantly higher than that of the control group (p<0.05), triglyceride (TG), low-density lipoproteins (LDL), high-density lipoproteins (HDL) and Apo-A1 levels were similar (p>0.05). An insertion/deletion (I/D) polymorphism with a significantly increased frequency was observed in the IUGR group (65%) as compared with the control group (33%) (p<0.05). When the distribution of the Apo-E gene polymorphism (E2, E3 and E4) was studied, no difference was found between the IUGR and control groups with respect to frequency. No Apo-B gene mutation was identified in the study groups. In conclusion, we may suggest that I/D polymorphism is responsible, though in part, for the etiology of intrauterine growth restriction. Levels of total cholesterol and Apo-B are elevated in IUGR infants, suggesting a linkage between low birth weight and atherosclerosis.

Methylenetetrahydrofolate reductase C677T and A1298C gene polymorphisms and therapy-related toxicity in children treated for acute lymphoblastic leukemia and non-Hodgkin lymphoma.

This study aimed to investigate the association of the methylenetetrahydrofolate reductase (MTHFR) gene C677T and A1298C polymorphisms with serum drug levels and toxicities after high-dose methotrexate (MTX) infusion. The study included 37 children with acute lymphoblastic leukemia or non-Hodgkin lymphoma. Serum MTX levels and toxicities of bone marrow, liver and kidney were analysed. Genotype analysis of the C677T and A1298C gene polymorphisms from genomic DNA of the subjects was performed by real-time PCR. Subjects with MTHFR polymorphism for C677T (CT, TT) had significantly higher MTX levels at 24 h (p = 0.009), and these genotypes did not seem to cause toxicity. Subjects with MTHFR polymorphism for A1298C (AC, CC) had significantly higher MTX levels at 48 h (p = 0.02), and had more grade III/IV anemia (p = 0.02), thrombocytopenia (p = 0.0001), elevated AST levels (p = 0.04) and frequent febrile neutropenic episodes (p = 0.004). The present study suggests that A1298C gene, but not C677T polymorphism is associated with MTX-related toxicity.

Absence of apolipoprotein B-3500 mutation in Turkish patients with coronary and cerebrovascular atherosclerosis.

OBJECTIVE: The arginine- to-glutamine change at codon 3500 of the apolipoprotein B-100 (apo B) is a well- known genetic cause of hypercholesterolemia. Since increased cholesterol levels lead to atherosclerosis, identification of the carries of the apo B-3500 mutation is an important step in the risk stratification of individuals and families with hypercholesterolemia. The prevalence of this mutation in Turkish population is not well known. We aimed to investigate the frequency of apo B-100 mutations (codon 3500) C9774T (Arg 3500-->Trp) and G9775A (Arg 3500-->Gln) in patients with atherosclerosis in comparison with healthy subjects. METHODS: This cross-sectional study included 442 unrelated subjects living on the West coast of Turkey. Subgroups consisted of 165 patients with coronary artery disease, 163 patients with ischemic stroke, and 114 healthy control subjects. RESULTS: We did not find any apo B-100 mutation both in the patient and control groups. CONCLUSION: As it is hypothesized that this mutation arose within the Central European region from a common ancestor approximately 7000 years ago and spread across Europe, our result of the absence of the R3500Q mutation in Turkish patients give an important information about the geographical distribution of the apo B-R3500Q, that the mutation has not reached to Anatolia.

Protein phosphatase 2A (PP2A) has a potential role in CAPE-induced apoptosis of CCRF-CEM cells via effecting human telomerase reverse transcriptase activity.

Caffeic acid phenethyl ester (CAPE) is one of the most effective components of propolis which is collected by honey bees. The aim of this study was to investigate the cytotoxic and apoptotic effects of CAPE in the CCRF-CEM cell line and to clarify the role of serine/threonine protein phosphatase 2A (PP2A) and human telomerase reverse transcriptase (hTERT) activity as an underlining mechanism of CAPE-induced apoptosis. Trypan blue dye exclusion test and XTT methods were used to evaluate the cytotoxicity and ELISA based oligonucleotide detection, which can be seen during apoptosis, was used to determine apoptosis. Acridine orange/ethidium bromide dye technique was also used to evaluate apoptosis. The cytotoxic effect of CAPE was detected in a dose and time dependent manner with the IC(50) of 1 muM. ELISA and acridine orange/ethidium bromide methods have shown remarkable apoptosis at 48th hour in CAPE treated cells. To investigate the role of PP2A in CAPE-induced apoptosis of CCRF-CEM cells, we performed combination studies with CAPE and, Calyculin A and Okadaic acid, which are very well known inhibitors of PP2A, in IC(20) of inhibitors and IC(50) of CAPE. Combination studies revealed synergistic effect of both drugs by concomitant use. Western blot analyses of PP2A catalytic and regulatory subunits showed down-regulation of expression of PP2A catalytic subunit in CAPE treated cells at 48th hour. Since, PP2A is important in hTERT (telomerase catalytic subunit) activation and deactivation, we also performed hTERT activity in CAPE treated cells simultaneously. Treating cells with IC(50) of CAPE for 96 h with the intervals of 24 h showed marked reduction of hTERT activity. The reduction of hTERT activity in CAPE treated CCRF-CEM cells was more prominent in the initial 48 h. The variation of hTERT activity in CAPE treated CCRF-CEM cells may be the reason for the protein phosphatase interaction that occurred after treatment with CAPE.

Prothrombin G20210A mutation in cases with recurrent miscarriage: a study of the mediterranean population.

BACKGROUND: Thrombophilic predisposition may be one of the underlying causes of recurrent miscariage (RM). The purpose of this study was to evaluate the Prothrombin G20210A mutation in cases with history of RM. MATERIAL AND METHODS: A total of 104 cases, 55 with diagnosis of RM and 49 control cases, were included in this controlled study. In all cases, in addition to full examination tests, Prothrombin 20210A mutation analysis was carried out by means of Polymerase Chain Reaction (PCR). RESULTS: Mean number of the abortion was 3.51 +/- 0.74 in the RM group and 0.08 +/- 0.27 in the control group (p < 0.05). As a consequence of comprehensive examinations, in 24 (43.6%) of 55 RM cases at least one etiologic factor was put forth. Prothrombin G20210A mutation was observed in six (10.9%) cases of the RM group and one (2.04%) in the control group (p < 0.05). Four of the six cases (66.7%) of Prothrombin G20210A mutation had a subsequent pregnancy. Among these four pregnancies, there was one spontaneous loss at 14 weeks of gestation and one severe pre-eclampsia. CONCLUSION: Our data together with literature suggest that Prothrombin G20210A mutation may be associated with RM. We recommend this genetic testing as a screening tool for women with history of RM.

The nature and origin of intraocular metallic foreign bodies appearing after phacoemulsification.

In order to evaluate the nature and origin of metallic foreign bodies embedded in the iris after uneventful phacoemulsification, we aimed to produce metallic fragments by applying ultrasound power with the same phaco machine and handpiece in an experimental model in vitro. In a glass bottle, we used linear phaco power of 100% continuously for 5 min first with a new phaco tip and then with a used tip. Afterwards, the fluid in the bottle was filtered through a Millipore filter. The remains on the filter were studied by scanning electron microscopy (SEM) and microprobe. Small particles remained on the filter which was used for the new tip. Microprobe microanalysis showed that these particles were mainly titanium, the same as the phaco tip. SEM of both the new and the used tips showed small fragments on the exterior surface and lumen. Intraocular metallic foreign bodies after phacoemulsification are likely to be shaken loose from the phaco tip. Although it is mostly agreed that these fragments are well tolerated, their overall effect remains to be evaluated in the long term.

Electrochemical genosensor based on colloidal gold nanoparticles for the detection of Factor V Leiden mutation using disposable pencil graphite electrodes.

Electrochemical genosensors for the detection of the Factor V Leiden mutation from polymerase chain reaction (PCR) amplicons using the oxidation signal of colloidal gold (Au) is described. A pencil graphite electrode (PGE) modified with target DNA, when hybridized with complementary probes conjugated to Au nanoparticles, responded with the appearance of a Au oxide wave at approximately +1.20 V. Specific probes were immobilized onto the Au nanoparticles in two different modes: (a) Inosine-substituted probes were covalently attached from their amino groups at the 5' end using N-(3-dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) as a coupling agent onto a carboxylate-terminated l-cysteine self-assembled monolayer (SAM) preformed on the Au nanoparticles, and (b) probes with a hexanethiol group at their 5' phosphate end formed a SAM on Au nanoparticles. The genosensor relies on the hybridization of the probes with their complementary targets, which are covalently immobilized at the PGE surface. Au-tagged 23-mer capture probes were challenged with the synthetic 23-mer target, 131-base single-stranded DNA or denatured 256-base polymerase chain reaction (PCR) amplicon. The appearance of the Au oxidation signal shortened the assay time and simplified the detection of the Factor V Leiden mutation from PCR amplified real samples. The discrimination between the homozygous and heterozygous mutations was also established by comparing the peak currents of the Au signals. Numerous factors affecting the hybridization and nonspecific binding events were optimized. The detection limit for the PCR amplicons was found to be as low as 0.78 fmol; thus, it is suitable for point-of-care applications.